Biochem. J. (1965) 94, 569

Induction and Multi-Sensitive End-Product Repressionin the Enzymic Pathway Degrading Mandelate in

Pseudomonas fluorescens

By J. MANDELSTAM AND G. A. JACOBY*National Institute for Medical Research, Mill Hill, London, N. W. 7

(Received 15 July 1964)

1. The first five enzymes involved in the degradation of mandelate in Pseudo-monas fluorescens have been examined. 2. Induction is not significantly affectedby glucose. 3. The first three enzymes form a group inducible by mandelate andrepressible by benzoate, catechol and succinate. 4. The pQssibility that benzoateand catechol act as repressors only after they have been degraded to succinate isunlikely since mutants blocked at suitable points in the pathway have the same

repression pattern as the wild type. 5. It is concluded that synthesis ofthe enzymesis subject to a multi-sensitive repression mechanism that can be independentlyactivated by benzoate or catechol or succinate. 6. In each case the repression can belargely overcome by increasing the concentration of the inducer. 7. The enzymes

of the first group are thus controlled by a dual system in which induction by thefirst substrate is opposed by repression exerted by the end product of the firstgroup and by the products of succeeding groups.

A previous study (McFall & Mandelstam, 1963a,b)on the behaviour of three enzymes in Eseherichiacoli suggested that the production of an induciblecatabolic enzyme is controlled not only by itsinducer but also by an end-product repression thatis specific. Thus ,B-galactosidase is induced bylactose, or other ,B-D-galactosides, and the inductionis prevented by galactose or some close metabolicderivative. Similarly L-tryptophanase and D-serine deaminase are each induced by their sub-strates and both are repressed by pyruvate, an endproduct of enzyme action in both instances. Othersystems have been described in which enzymesynthesis is controlled by induction and by end-product repression, e.g. nitrate reductase inChlorella vulgaris (Morris & Syrett, 1963), amidasein Pseudomonas aeruginosa (Brammar & Clarke,1963) and glucosamine 6-phosphate deaminase inBacillus subtilis (Clarke & Pasternak, 1962).The present paper describes the interaction of

induction and end-product repression in a longerand more complex pathway, the series of enzymesthat degrades mandelic acid in Pseudomonasfluorescens. This pathway is as follows:

E1 E2Mandelate -* benzoylformate --

E3.and ESb E4 Esbenzaldehyde -> benzoate -+ catechol --*--,-oxoadipate -> succinyl-CoA + acetyl-CoA

* Present address: Massachusetts General Hospital,Boston, Mass., U.S.A.

This system was extensively investigated byStanier and his colleagues (see Stanier, 1951) and isthe classic example of 'sequential induction', sincethe addition of any intermediate caused thesuccessively induced formation of all the ensuingenzymes in the pathway. Stanier, Hegeman &Ornston (1964) have since provided evidence thatenzymes E1, E2, E3a and E3b form a single groupapparently controlled by an operon, inducedsimultaneously and equally well by mandelate orbenzoylformate. The benzoic acid producedby the action of this group of enzymes theninduces E4, which appears to be separatelycontrolled operon. The catechol formed by E4then induces pyrocatechase (Es) and the otherenzymes leading to f-oxoadipate. These enzymesform a third group. According to this later workthe pathway is thus still sequentially induced,but the number of steps is less than was originallythought. The criteria of an operon in this systemare physiological (Stanier et al. 1964) only, andthere is as yet no genetic evidence that the genesdetermining the structures of these enzymes areclosely linked.The present work, which has been published in a

preliminary form (Mandelstam, 1964a,b,c), dealswith the first five enzymes and points to the con-clusion that the enzymes of the first operon aresubject not only to induction but to repression bythe end product resulting from the action of thatgroup of enzymes and independently by the endproducts of each succeeding group.

569

J. MANDELSTAM AND G. A. JACOBY

METHODS

Organism. Pseudomonas fluorescens A3.12 (obtained fromDr R. Y. Stanier) was used. It was grown with shaking at300 in a basal mineral salts medium containing (per 1.):KH2PO4, 13-6 g.; NH4C1, 1-0 g.; (NH4)2SO4, 1-0 g.;

MgSO4, 20 mg.; Fe(NH4)2(SO4)2,6H20, 1-56 mg. The pHwas adjusted to pH 7 with NaOH. Unless otherwise stated,the carbon source was glucose (5 mg./ml.). The bacteriawere washed with a solution containing KH2PO4 (13-6 g./l.)adjusted with NaOH to pH 7.

Isolation of mutants. Mutants defective in one of theenzymes in the sequence were obtained as follows. Cellsfrom an exponential culture were suspended in 0 2 M-acetatebuffer, pH 5-0, at a density of 3 mg. dry wt./ml., and to4 ml. of this was added 1-0 ml. ofa solution ofN-methyl-N'-nitro-N-nitrosoguanidine (4 mg./ml.). After incubation at350 for 30 min. the suspension was centrifuged, the cells

were washed once with phosphate buffer (10 ml.) andsuspended in 250 ml. of broth medium (hydrolysed caseinplus yeast extract). The culture was divided into fiveequal portions and left to grow overnight at room tem-perature. The cells were subcultured twice in basalmedium with sodium succinate (5 mg./ml.) as carbonsource. Inocula (1000-2000 cells/plate) were plated on

basal medium containing mandelic acid (250 jig./ml.)solidified with agar (1-5 g./100 ml.). Wild-type bacteriaproduced colonies of very uniform size after incubation for24 hr. at 35°. Plates were then left at room temperature(18-20°) for a further 3-6 days. Mutants, blocked in thepathway and therefore unable to utilize mandelic acid,then developed as pin-point colonies readily distinguishedfrom the wild type under a low-power microscope. Thesemutants were picked and tested for ability to utilize theintermediates of the pathway as growth substrates. Whenthe position of the block had thus been determined, it wasquantitatively checked, by inducing with mandelate a

culture growing in glucose medium (see below) and measur-

ing the enzyme in question. Those mutants that were

'leaky' (i.e. that had low activity after induction by growthin the presence of mandelate) were discarded. The mutantsretained for use had no measurable enzymic activity andfailed to show growth on the substrate in question for atleast 24 hr.

Induction in growing cultures. Except in the preliminaryexperiments (see below) induction was carried out with cellsin exponential-growth phase (0-lmg./ml.) in glucose mediumwith inducer at 250 pg./ml., except when stated otherwise.Samples for assay were treated as described below.Oxygen uptake by whole cells. Samples (10 ml.) were

centrifuged, washed and resuspended in phosphate buffer(6 ml.). Portions (2-5 ml.) were assayed by conventionalmanometric methods with the substrates at the followingconcentrations: mandelic acid, 2-5 mg./ml.; benzoic acid,2-5 mg./ml.; catechol, 300 jg./ml. Values were correctedfor endogenous respiration, and are expressed in units of1tmoles of 02/min.El: mandelate dehydrogenazse. Samples (100 ml.) were

centrifuged, washed once with buffer, resuspended in8-5 ml. of 0-9% NaCl solution and disrupted by ultrasonicvibration in an oscillator (25 keyc./see.; 250 w) for 3 min.Unbroken cells were removed by centrifuging (2500g for10 min.) and the enzyme was determined as described byStanier (1955).

E2: benzoylformate decarboxylase (benzoylformate carboxy.lyase, E0 4.1.1.7). In some experiments the enzyme wasdetermined with a portion (1-0 ml.) of the cell-free extractobtained as above, after ultrasonic disruption (see Stanier,1955). However, similar values were more simply obtainedwith toluene-treated samples prepared as follows. Portions(10 ml.) were centrifuged and the cells washed and re-suspended in 6 ml. of 0-1 M-phosphate buffer, pH 6-0,containing thiamine pyrophosphate (100 ,g./ml.). Twodrops of toluene were added, and the tubes stoppered,shaken vigorously and left for 30 min. Toluene was drivenoff by blowing air through the suspension for about 2 min.,and 2-5 ml. portions were taken for manometric determina-tion of the CO2 produced anaerobically after the additionof benzoylformate (final concn. 1 mg./ml.). Values werecorrected for CO2 retention.EV: benzaldehyde dehydrogenase (NAD+-linked) (benz-

aldehyde-NAD oxidoreducta-se, EC 1.2.1.6). When attemptswere made to measure this enzyme in ultrasonically dis-rupted preparations by following the reduction of NAD+(Stanier, 1955), values were erratic and frequently noactivity was detectable even with extracts of fully adaptedcells. The reason was found to be the presence, in cellsgrown under our conditions, of a potent NADH oxidase.The interfering activity was abolished in the presence ofNaCN (final conen. 100,ug./ml.). The procedure finallyadopted was as follows. The following additions were made,in the order given, to a spectrophotometer cell: 1-2 ml. of1-0 M-phosphate buffer, pH 8-5; 0-3 ml. of NaCN solution(1 mg./ml.); 1-0 ml. of cell-free extract (equivalent to about2 mg. dry wt. of bacteria); 0-3 ml. ofNAD+ solution (1 mg./ml.). The reaction was started by adding 0-2 ml. of aqueousbenzaldehyde (1-0 mg./ml.). The change in extinction at340 m,t was measured at 15 or 30 sec. intervals against ablank containing water instead of benzaldehyde.

E3b: benzaldehyde dehydrogenase (NADP-linked). Thisenzyme was not investigated.

EV: benzoate oxidase. No method was found of breakingthe cells without losing most or all of the activity. This is inkeeping with the findings of other workers.

Es: pyrocatecha8e. The enzyme was estimated mano-metrically (Hayaishi, Katagiri & Rothberg, 1957) with cell-free extracts prepared as above by ultrasonic disruption.Enzyme unit8. All units are expressed as m,tmoles of

substrate destroyed/min.; specific activities are units/mg.dry wt. of bacteria.

Reagent8. Benzoylformic acid was synthesized by themethod of Oakwood & Weisgerber (1955); NAD+ (90%pure) was obtained from British Drug Houses Ltd., Poole,Dorset.

RESULTS

Effect of glucose on induction of mandelate pathwayin non-growing cells. Before carrying out inductionexperiments in growing cultures it was necessaryto find a source of carbon that would not act as arepressor. Succinate, which is a useful non-repressing carbon source for fl-galactosidase inE. coli, was found to be a strong repressor inPseudomonas (see below).

Glucose was then tested for repressor activity onthe mandelate-pathway enzymes in non-growingcells. In other systems the non-growing state

570 1965

INDUCTION AND REPRESSION IN PSEUDOMONAS

causes maximum accumulation of carbon-sourcerepressors (see Mandelstam, 1961), and it wastherefore expected that any repressor action ofglucose would be most strongly expressed underthese conditions.

Cells in exponential growth in glucose-minimalmedium were harvested, washed, suspended inphosphate buffer at 0-5 mg. dry wt./ml. and inducedwith mandelic acid (5 mg./ml.) in the absence andin the presence of glucose (5 mg./ml.). Samples(10 ml.) were taken at intervals and the ability ofthe whole cells to oxidize mandelic acid wasmeasured. Under these conditions, not only didglucose fail to repress, but it actually caused afivefold stimulation in the rate of formation ofmandelate-pathway enzymes as measured byoxidative capacity (Fig. 1).

Timie (min.)

Fig. 1. Effect ofglucose on adaptation to mandelate in non-growing cells. Cells were suspended in phosphate buffer at0-5 mg. dry wt./ml. with mandelate alone (1 mg./ml.) (0)or with glucose (5 ml./ml.) (o) in addition. The suspensionswere shaken at 300 and samples removed. The ability ofwashed whole cells to oxidize mandelate was measured.

In growing cultures there was again no strongrepressor action of glucose. Cultures grown for3-5 hr. in the presence of mandelate (2 mg./ml.)together with glucose (5 mg./ml.) had 80-100% ofthe enzymic activity of cultures grown withmandelate alone. Under comparable conditionsthe inducible systems that degrade amino acids inthe same strain of bacteria show a much moremarked glucose effect (Jacoby, 1964).

Since glucose was relatively non-repressive forthis enzyme sequence, it was subsequently used asa carbon source, and the remaining experimentsdescribed below were done with growing cultures.

End-product repre.sion8 in the mandelate path-way. A series of experiments was done in which,with mandelate as inducer, subsequent metaboliteswere tested in turn as repressors. The experimentswere all done with cultures growing with glucose ascarbon source and with inducers and repressorsadded as indicated. At the concentrations usednone of these substances inhibited growth, butsuccinate (see below) caused some stimulation. Theresults can be most easily appraised by consideringfirst the effects these substances have on thesubsequent ability of whole cells to oxidize mandel-ate or benzoate. It was shown (see below) thatoxidation by whole cells is, in fact, a reliablereflexion of what is happening to the individualenzymes.

Benzoylformate had no significant repressoractivity. Benzaldehyde was not initially tested, butsubsequent work showed that it did repress man-delate dehydrogenase (I. L. Stevenson & J. Mandel-stam, unpublished work). Benzoate in the presenceof mandelate repressed mandelate-oxidative capa-city by about 80-90% but induced benzoate-oxidative capacity as well as mandelate did, andoccasionally better. Catechol repressed bothactivities. A typical result is shown in Table 1.The findings suggested that benzoate was

repressing some or all of the preceding enzymes(i.e. E1-E3) but as originally suggested (see Stanier,1951), inducing E4 and subsequent enzymes,

Table 1. Effect of benzoate and catechol on induction of the capacity of whole cellBto oxidize mandelate and benzoate

A culture growing in glucose medium was treated with mandelate alone or with mandelate in the presence ofbenzoate or catechol. Inducer and repressor concentrations were 250 ,ug./ml. After one generation samples weretaken and the oxidative capacity of washed whole cells was measured with either mandelate or benzoate assubstrate.

Induction medium

MandelateMandelate+ benzoateMandelate+ catechol

Mandelate oxidationA

RepressionUnits/mg. (%)

27025 9110 96

Benzoate oxidation

RepressionUnits/mg. (%)

142 -106 256 96

Vol. 94 571

J .MANDELSTAM AND G. A. JACOBYTable 2. Repression of enzymes in the mandetate pathway by benzoate

Bacteria were grown in glucose medium with mandelate (250 ,ug./ml.) orwith mandelate+ benzoate (2501,ug./ml.).The culture density increased in both cases from 0 09 to 0-18 mg./ml. Enzyme activities were determined inultrasonically disrupted cells (see the Methods section). Values are specific activities expressed as units/mg. dry wt.of bacteria before disruption.

Sp. activity (units/mg. dry wt.)

EnzymeInduction medium...... Mandelate

E1 (mandelate dehydrogenase)E2 (benzoylformate decarboxylase)E3a (benzaldehyde dehydrogenase)E4* (benzoate oxidation)E5 (pyrocatechase)

13568538127193

Mandelate+Benzoate

10419.4

130198

Repression (%)92947500

* E4 activity is lost during ultrasonic disruption: values refer to 02 uptake by whole cells (mfimoles/min./mg. dry wt.).

Table 3. Repression of enzymes in the mandelate pathway by catechol

The experiment was done as described in Table 2 except that benzoate was replaced by catechol (250 ,tg./ml.).Sp. activity (units/mg. dry wt.)

Enzyme Mandelate+Induction medium...... Mandelate catechol

E1 (mandelate dehydrogenase) 108 25E2 (benzoylformate decarboxylase) 420 143E3 (benzaldehyde dehydrogenase) 34 16E4* (benzoate oxidation) 120 7E5 (pyrocatechase) 123 135

* 02 uptake by whole cells (see footnote to Table 2).t Negative value indicates stimulation.

Repression (%)77665394

(- 10)t

whereas catechol was repressing all the precedingenzymes including E4.The effects of benzoate on the formation of

individual enzymes were confirmed by disruptingthe cells after induction and measuring the enzymicactivities of the cell-free preparation. The firstthree enzymes, i.e. those preceding benzoate in thepathway, were all repressed by about 75-95%(Table 2). E5 (pyrocatechase), on the other hand,was fully induced, and E4, though the measure-ments had to be made with whole cells, alsoappeared to be fully induced.The effect of catechol on the production of the

same enzymes is shown in Table 3. E1, E2 and E3were repressed 50-80%, whereas E5 was fullyinduced. The fact that E5 was present in highamounts indicates that the low values for benzoateoxidation by cells grown in the presence of catechol(see also Table 1) were due to repression of E4.The results thus far may be summarized as

follows. Benzoate represses E1, E2 and E3, andinduces first E4, and then, as would be expected onthe basis of sequential induction, E5 and the sub-sequent enzymes: catechol represses E1-E4, but

induces E5 and the subsequent enzymes (again assuggested by Stanier, 1951).An attempt was made to determine whether

f-oxoadipate, the end product of the group ofenzymes beginning at E5, acted as a repressor, butthis substance was apparently unable to enter thecells. Organisms adapted to growth on mandelate,and therefore presumably adapted to f-oxoadipate,failed to utilize it as a carbon source for growth,and showed no significant oxidation when testedmanometrically.

Suceinate, as a final product of the pathway, wasalso tested. This usually produced a somewhatweaker repression (40-60%) of E1, E2 and E3, buthad more effect on the synthesis of E5 (about 85%repression). A typical result is shown in Table 4.Oxidation of benzoate by whole cells was also re-pressed but, since E5 activity was also low, it cannotbe categorically stated that E4 was repressed.Repressor effects produced by succinate were morevariable than those produced by benzoate orcatechol, and repression of E1-E3 was occasionallymuch greater than the result shown in Table 4.

Acetate, when tested as a repressor, had much the

1965572

INDUCTION AND REPRESSION IN PSEUDOMONAS

Table 4. Repression of enzymes in the mandelate pathway by succinate

The experiment was done as described in Table 2 except that benzoate was replaced by succinate (500 tug./ml.).Sp. activity (units/mg. dry wt.)

EnzymeInduction medium......

E1 (mandelate dehydrogenase)E2 (benzoylformate decarboxylase)E3 (benzaldehyde dehydrogenase)E4* (benzoate oxidation)E5 (pyrocatechase)

Mandelate+Mandelate succinate

98 43374 15430 1692 35118 18

* 02 uptake by whole cells (see footnote to Table 2).

same effect as succinate, i.e. it appeared to repressall the activities El-E5. Other substances were notinvestigated, but presumably any intermediate inthe tricarboxylic acid cycle or anything contribut-ing material to the cycle would tend to produceeffects resembling those of succinate.

Specificity of repression. Since the repressors, atthe concentrations used, did not affect growth, itwas apparent that they were not general inhibitorsof protein synthesis, but a more detailed examina-tion of their specificity was clearly desirable. Theireffect was accordingly examined on the formation ofthe induced enzyme sequence oxidizing p-hydroxy-benzoate. This pathway converges with the mandel-ate pathway at ,B-oxoadipate. A culture in glucosemedium was allowed to double in the presence ofp-hydroxybenzoate (250 ,ug./ml.) and the substancebeing tested as a repressor (also at 250 ,tg./ml., butat 500 ,ig./ml. for succinate). Samples were takenand the oxygen uptake was measured with p-

hydroxybenzoate as substrate.There were obvious differences in the repression

patterns of the two pathways. Benzoate, a very

potent repressor for the mandelate-pathway en-

zymes, had no effect on adaptation to p-hydroxy-benzoate, and catechol acted only as a weak repres-sor in the latter system (giving less than 50%repression). Acetate, which inhibited mandelateadaptation by about 90%, decreased adaptation top-hydroxybenzoate by only about 30O%. Succinate,however, affected both systems more or less equally.

It seems reasonable to conclude that the re-

pressor effects, at any rate those produced bybenzoate and catechol, were specific and were notthe result of an effect on enzyme induction ingeneral.

Possible modes of repressor action. Consideringfor the moment the repression of enzymes of thefirst operon by benzoate, catechol and succinate,the following explanations seem possible:

(a) Multi-sensitive repression system. The re-

pression mechanism is sensitive to, and can be

independently activated by, each of the repressor

substances.(b) Repression by one end product. There is only

one repressor (e.g. succinate itself or some com-

pound closely related to it metabolically), andbenzoate and catechol are repressors because theyare both converted into it. This possibility was

ruled out by the use of suitable mutants. Thus a

strain benz- (unable to form benzoate-oxidizingenzyme) was isolated and induced with mandelicacid in the presence and absence of benzoate. Inthis organism benzoate repressed the developmentof mandelate-oxidative capacity, and catechol andsuccinate were also repressors (Fig. 2), and theresults did not differ significantly from those foundwith the wild type. The effect of benzoate on theformation of benzoylformate decarboxylase was

checked and the results were qualitatively similarto those shown in Fig. 2.Although the benz- mutant had no measurable

benzoate-oxidase activity, the possibility had to beconsidered that it was 'leaky' and that an amountof enzyme too small to be measured was neverthe-less providing a trickle of succinate that was thecause of the repression. One would then expectthat, if benzoate caused repression only afterdegradation to succinate, the addition of benzoateto a culture containing an excess of succinateshould produce no further effect. An experimentwas accordingly carried out in which succinate at1 mg./ml. (i.e. twice the concentration usedpreviously) and benzoate (250 ,ug./ml.) were testedseparately and together. In the presence of bothsubstances the repression was greater than thatwith either alone (Fig. 3).The experiments with the benz- mutant suggest

that benzoate and succinate act separately as

repressors and that their effects are additive.A number of mutants were also obtained that

were blocked between catechol and fl-oxoadipate.These mutants displayed the same effects as thebenz- and wild type, i.e. catechol remained a

Repression (%)5659476285

573Vol. 94

J. MANDELSTAM AND 0. A. JACOBY

150

1-1

'C)I2..

0

-4

4)40-E

0

r4)

50

R4

0 0 025 0 05

Increase in bacterial mass (mg./ml.)

Fig. 2. Effect of benzoate, catechol and succinate on

adaptation to mandelate in a benz- mutant. A mutantblocked at E4 (benzoate oxidation) was used. Cells growingexponentially in glucose-minimal medium were inducedwith mandelate (250 pg./ml.) alone (o) or with one of thefollowing present as well: benzoate (250 ,ug./ml.) (-),catechol (250 ,ug./ml.) (A), or succinate (500 ,ug./ml.)(A). Samples were taken at 20 min. intervals for hr. andthe ability of washed whole cells to oxidize mandelate was

measured.

potent repressor though it could not be convertedinto succinate. Benzoate and succinate, as

expected, were still repressors in this strain.(c) 'Sequential repression.' On this hypothesis

each group of enzymes in the pathway is repressedonly by the immediate end product of action of theenzymes of that group. Thus if we assume, as

suggested by Stanier et al. (1964), that E1-E3comprise a co-ordinated group, then benzoate is itsrepressor. The second operon, determining E4, isrepressed by catechol. The third operon, deter-mining E5, and the next few enzymes are re-

pressed by ,-oxoadipate, and so on until succinaterepresses the last enzymes ofthe induced pathway.If, then, cells are induced by adding mandelate,some induction will occur and material will beginto flow down the pathway. If succinate has alsobeen introduced into the medium it will begin byrepressing the last enzymes of the sequence, thusleading to accumulation of f-oxoadipate. This will,

.4)

1~-

.4

0

~0

4)

PCi

41)

; 200C)0

0

Z300

4)4

= 200 /.

~ioo

0 005 0-10Increase in bacterial mass (mg./ml.)

Fig. 3. Additive repression produced by benzoate andsuccinate in a benz- mutant. For procedure see the legendto Fig. 2. Additions were as follows: 0, mandelate (250 ,g.jml.) alone; *, mandelate+benzoate (250 ,ug.fml.); A,

mandelate+succinate (1 mg./ml.); U, mandelate+ben-zoate+ succinate.

0 0-025 0 05 0.075

Increase in bacterial mass (mg./ml.)

Fig. 4. Repression of benzoylformate decarboxylase (E2)in an md- mutant. The mutant was blocked at E1 (man-delate dehydrogenase). Cells growing exponentially inglucose-minimal medium were treated with mandelate,which acts as an inducer for E2 and E3 (see Stanier et al.1964). Samples were taken at 20 min. intervals for 1 hr.and the decarboxylase was measured in toluene-treatedpreparations (see the Methods section). Additions were as

follows: o, mandelate (250 ,ug.fml.); *, mandelate+benzoate (250 ,tg./ml.); A, mandelate+ catechol (250 jtg./ml.); E, mandelate+ succinate (500 jug./ml.).

in tum, repress the enzymes from E5 onwards so

that catechol will accumulate etc. etc., until finallythe enzymes of the first operon are repressed.

574 1965

INDUCTION AND REPRESSION IN PSEUDOMONAS

- 3002

cz4-

C.

0

SM

= 200

SSC.)

i-~

1 oo I

0 0-05 0-10Increase in bacterial mass (mg./ml.)

Fig. 5. Effect of inducer concentration on repressionproduced by succinate. Wild-type cells growing in glucose-minimal medium were treated with succinate (500 ,ug./ml.)as repressor and with various amounts of inducer, as

follows: e, mandelate (250 ,ug./ml.); A, mandelate (500 pg./ml.); *, mandelate (1000 ,ug./ml.); o, control culturecontaining no succinate and induced with mandelate at250 jig./ml.

The possibility was ruled out by the use of a

mutant md4 (lacking mandelate hydrogenase).It was treated with mandelate, and the observa-tion of Stanier et al. (1964) was confirmed thatthis caused induced formation of benzoylformatedecarboxylase. In this mutant material does notpass down the pathway because of the block at El;nevertheless, benzoate and the other two repressorswere still active (Fig. 4) and the results againresembled those obtained with the wild type.The elimination of possibilities (b) and (c) leaves

(a) as the most likely explanation.Reversibility of repre8ssion. With f-galactosidase

in E. coli the repression produced by metabolicproducts could not be reversed by inducer (Mandel-stam, 1962).The reversibility of end-product repression in

the mandelate pathway was examined as follows.

400 0

300

200

0

0 0 025 0 05 0*075Increase in bacterial mass (mg. ml.)

Fig. 6. Effect of inducer concentration on repressionproduced by benzoate. Details are as in Fig. 5, withbenzoate (250,ug./ml.) in place of succinate.

Several flasks of culture were set up with the usualglucose medium and a fixed concentration (500 ,ug./ml.) of succinate as repressor. Inducer (mandelate)was added at concentrations varying from 250to 1000 or, occasionally, 2000,ug./ ml. (Prelimin-ary experiments had shown that the lowest concen-tration of inducer was sufficient to give a maximumrate of induction when no succinate was present.)The repression resulting from the presence ofsuccinate was readily reversed by increasing theconcentration of inducer (Fig. 5).The repression induced by benzoate (250 ,pg./ml.)

or by catechol (125 ,ug./ml.) was also reversed byincreasing the amount of inducer. An experimentwith benzoate is illustrated in Fig. 6.

In case the measurements of oxidation by wholecells might be misleading, some of the experimentswere checked by using mandelate as inducer andmeasuring the benzoylformate decarboxylase intoluene-treated preparations. The results weresimilar to those obtained with whole cells.

DISCUSSION

Reference has already been made (see theintroduction) to the fact that, in E. coli, the produc-tion of fl-galactosidase, tryptophanase and D-serinedeaminase appears in each case to be jointlydetermined by induction and by end-productrepression. In all three a single metabolic stepdegrades the inducing substrate to one of thecommon intermediary metabolites of the cell:galactose in the first case and pyruvate in the othertwo. One of the reasons for choosing the mandelate

Vol. 94 575

J. MANDELSTAM AND G. A. JACOBY

pathway is that a sequence of about eight enzymesis involved. This is roughly the number of enzymesconcerned in biosynthetic sequences producingarginine or histidine, which are all controlled byend-product repression. Though the mandelatepathway also has this property, its regulationpresents a number of features that distinguish itfrom other pathways, both catabolic and bio-synthetic.An obvious difference between the P-galactosi-

dase ofE. coli and the mandelate-pathway enzymesis that, in the former, the effects of metabolicrepression could not be reversed by inducer(Mandelstam, 1962). This suggested that themetabolic repressor was independent of therepressor postulated by Jacob & Monod (1961) asthe cause of inducibility. Supporting evidence wasthe occurrence of metabolic repression in mutanttypes (Oc or i-) that are constitutive for P-galacto-sidase (Brown, 1961; Mandelstam, 1962). Thesemutants have been postulated to be either unable tomake functional repressor (i-) or to be largelyinsensitive to its action (oc), so that the enzyme isproduced constitutively.

These facts led to the conclusion (Mandelstam,1962) that the formation of f-galactosidase iscontrolled by two distinct types of repressor, one ofwhich is controlled by the i gene whereas the otheris formed during the metabolism of carbon com-pounds. The second repressor was tentativelyidentified as galactose or some metabolicallyrelated compound (McFall & Mandelstam, 1963b).The case for the separate existence oftwo repressorsfor the fl-galactosidase system ofE. coli is supportedby the observations of Loomis & Magasanik (1964).

In the mandelate-pathway enzymes the repres-sion of the first operon, whether produced bybenzoate, catechol or succinate, is readily reversedby the inducer. A strict demonstration of com-petitive interaction is not possible since both theinducer and the repressors are being metabolized.However, the mere fact that there is interactionbetween inducer and repressor raises the possibilitythat they may be acting at the same site. Gorini(1963) has described an analogous reaction in thesystem of enzymes synthesizing arginine fromglutamate in E. coli. The whole sequence isrepressed by the end product, arginine, and therepression is reversed, more or less competitively,by glutamate, the first substrate. This interactionbetween the first and last substrates is formallyanalogous to that found in the mandelate pathway.Both instances suggest that these control mechan-isms may differ substantially from those governingthe formation of the lactose system of E. coli.A distinctive property of the mandelate system is

the repression of enzymes of the first operon by atleast three separate substances: (a) benzoate, (b)

catechol (or one of the immediately succeedingmetabolites) and (c) succinate (or some relatedmetabolite). The possibility of a 'sequentialrepression' (see the Methods section) seems to beruled out. Another possibility, that benzoate andcatechol repress only after being degraded tosuccinate, is equally unlikely in view of the fact thatmutants blocked at E4 and E5 exhibit the samerepression pattern as the wild-type parent. Theremaining explanation is that formation ofenzymesof the first operon is governed by a multi-sensitiverepression mechanism that can be activatedindependently by at least three metabolites in thepathway. We have introduced the term 'multi-sensitive' repression to describe this mechanismand to distinguish it from the 'multivalent'repression system found in coliform bacteria (seeUmbarger, 1964). (In the latter a number ofenzymes supplying intermediates used in thesynthesis of the branched amino acids are onlyrepressed when all the end products are presenttogether in excess.)The experiments described in the present paper

in no way indicate the site of repressor action, andthe possibilities are as follows:

(a) Effect on transport of inducer into the cell,i.e. the repressors inhibit the functioning of aspecific permease. This would imply the existenceof a permease molecule with specific sites for the'substrate' (mandelate) and for each of the repres-sors, that is an 'allosteric' molecule (see Monod,Changeux & Jacob, 1963) with at least four specificcombining sites.

(b) An effect on the protein-synthesizing mechan-ism. There are a number of possible sites where arepressor might act: at the gene level, as suggestedby Jacob & Monod (1961), or at the ribosome level,as suggested by Hauge, MacQuillan, Cline &Halvorson (1961). In either case, if we assume thatthe inducer and the repressors act on the samemolecule, it is necessary to postulate that thismolecule has at least five recognition sites: one forthe inducer, one for each of three repressors and onefor the relevant portion ofDNA or messenger RNAwhere it is supposed to act. The alternative is topostulate a rather non-specific site with affinity forrepressors of widely differing chemical character-istics.

(c) Finally, the possibility must be consideredthat the inducer and the repressors do not all reactwith the same molecule.

The experimental resolution of this question willprobably not be possible until a reliable cell-freesystem for forming the induced enzymes becomesavailable.The advantage to the cell, in terms of protein

economy, of having control by induction and byend-product repression has been discussed by

576 1965

Vol. 94 INDUCTION AND REPRESSION IN PSEUDOMONAS 577

S2

I 4]

j E4}

S5

EsI E

,B-Oxoadipate~1'Succinate

Scheme 1. Representation of induction and repression inthe mandelate pathway. Mandelate (Si) is degraded by aseries of enzymes E1 etc. to the tricarboxylic cycle. E1-E3are determined by an operon and are inducible by Si; E4 isindependently inducible by benzoate; E5 and the ensuingenzymes before /-oxoadipate are determined by a thirdoperon inducible by catechol (see Stanier et al. 1964).The enzymes of the first operon are repressed byS4 (the end product to which they give rise), byS5 (the end product of the second group) and alsoby succinate (/3-oxoadipate may also be a repressor,but it fails to enter cells and thus cannot be tested). Therepressors act additively, and their effect can be counter-acted by increasing the concentration of Si. Si, mandelate;S2, benzoylformate; S3, benzaldehyde; S4, benzoate;Ss, catechol.

McFall & Mandelstam (1963a). The mandelatepathway exhibits the same general property but ina much more elaborate form (Scheme 1). Thiscomplexity may have been evolved to deal with theintricate system of converging pathways by whichthe pseudomonads can degrade a large variety ofaromatic carbon compounds to components of thetricarboxylic acid cycle.

This work was done during the tenure of a U.S. PublicHealth Service Fellowship (no. EPD-18.124) awarded toG. A. J. by the National Institute of Allergy and InfectiousDiseases. We are indebted to Miss Barbara Alkins andMiss Gillian Holloway for technical help.

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